Characterization of Streptococcus equi subsp. zooepidemicus isolates containing lnuB gene responsible for the L phenotype

Within the framework of the β-hemolytic streptococci surveillance carried out by the National Reference Laboratory from Uruguay, three putative Streptococcus equi subsp. zooepidemicus (SEZ) were received from different health centers. Being these the first reports associated with human infections in Uruguay, the objective of this work was to confirm their identification, to determine their genetic relationship and to study their antibiotic susceptibility. Using four different methods, they were identified as SEZ, a subspecies which has been described as the etiologic agent of rare and severe zoonosis in a few cases in other countries. The three isolates presented different pulsotypes by PFGE; however, two of them appeared to be related and were confirmed as ST431 by MLST, while the remaining isolate displayed ST72. Their resistance profile exhibited an unexpected feature: despite all of them were susceptible to macrolides, they showed different levels of resistance to clindamycin, i.e. they had the so-called “L phenotype”. This rare trait is known to be due to a nucleotidyl-transferase, encoded by genes of the lnu family. Although this phenotype was previously described in a few SEZ isolates, its genetic basis has not been studied yet. This was now analyzed by PCR in the three isolates and they were found to contain a lnuB gene. The lnuB sequence was identical among the three isolates and with many lnuB sequences deposited in data banks. In conclusion, for the first time in Uruguay, three SEZ isolates recovered from non-epidemiologically related cases of human invasive infection were identified. Moreover, this is the first report about the presence of a lnu gene in the S. equi species, revealing the active lateral spread of the lnuB in a new streptococcal host.


Bacterial isolates
Within the framework of the β-hemolytic streptococci surveillance, carried out by the National Reference Laboratory of the Uruguayan Ministry of Public Health, we regularly receive isolates followed by demographic information about the patients. Our routine protocol implies the assignment of a lab number to identify each isolate. After that, the patient information is anonymized and de-identified prior to analysis. This surveillance is approved by Dirección General de Salud, Ministerio de Salud Pública. Between 2018 and 2019, three putative Streptococcus equi subsp. zooepidemicus (SEZ) from different health care providers were received at the NRL. Patients were elderly people between 75 and 90 years old and all of them had been in contact with domestic or farm animals. The first isolate (SEZ 559) was recovered from the cerebrospinal fluid of a patient who developed fatal meningitis. The second isolate (SEZ 567) was obtained from the blood of a patient who had fever of unknown origin. The third isolate (SEZ 594) was recovered from the blood of a patient with pneumonia. The last two patients had a good outcome. The food consumption habits of the three patients were unknown.

Detection of lincosamide and tetracycline resistance genes
A PCR approach was carried out to look for the lnu gene in the three isolates. First, the five lnu genes (lnuA, B, C, D and E) present in gram positive bacteria were searched for in data banks up to June, 2021. A representative sequence of the five lnu genes was chosen to conduct a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). These correspond to the following accession numbers: CP038184.1 for lnuA, CP031556.1 for lnuB, CP021772.1 for lnuC, CP033165.1 for lnuD and KF287643.1 for lnuE. No release of Streptococcus equi was found to contain the lnu sequences, or even traces of them. Taking into account that the lnuB gene is the most broadly distributed of lnu genes [19,26,27,29,31], the analysis was focused on this gene. Among streptococci, no matches were found in group C-Lancefield members, but six S. agalactiae strains resulted positive (ACs: CP031556, CP021773, CP021862, CP058666, CP059383 and KF772204). Considering that, among lnuB positive strains found in the public data banks, S. agalactiae was the most closely related species to SEZ, these six lnuB sequences were employed to design a lnuB primer pair. Specifically, forward and reverse primers were designed to amplify a DNA segment containing the entire 804 bp-gene, plus 16 bp upstream the start codon and ten bp downstream the stop codon (Table 1). S. agalactiae M6390 was taken as a reference of a lnuB positive strain (AC: HM209466) [31]. This strain was received at the NLR as part of the Latin American Program for Quality Assurance in Bacteriology and Antimicrobial Resistance (LA-EQAS), coordinated by the Pan American Health Organization and the Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr. Carlos G. Malbrán". In the case of tetracycline resistance, the presence of tetM and tetO determinants were assayed by PCR using oligonucleotides previously described (Table 1) [32].
To prepare templates for PCR reactions, genomic DNAs were extracted with the "Genomic DNA Purification kit" (Thermo Scientific, #K0512). PCR reactions were performed in a total volume of 30 μL containing 1X buffer (BIORON), 200 μM of each deoxynucleotide triphosphate (Fermentas), 300 nM of each primer (Macrogen), 2U of High Taq polymerase (BIORON) and template DNA (100 ng and 800 ng for tet and lnuB amplifications, respectively). The conditions used were: 2 min at 94˚C, followed by 30 cycles of 94˚C for 30 s, 30 s at the annealing temperature, 1 min at 72˚C, and a final extension step at 72˚C for 5 min. Amplicons were purified with the MiniElute purification kit (Qiagen, #28006) and sequenced at Macrogen Inc. (Seoul, Korea).
Nucleotide sequences of the lnuB gene harbored by SEZ 559, SEZ 567 and SEZ 594 strains have been deposited at GenBank under the accession numbers OP355463, OP355464, OP355465, respectively.

Pulsed-field gel electrophoresis (PFGE)
For PFGE analysis each isolate was collected with a swab from a fresh plate of overnight growth and suspended in 2 mL of PIV buffer (1.0 M NaCl in 10 mM Tris-HCl, pH 8). Then, the absorbance was measured using 1 mL at 578 nm and adjusted between 2.0 and 3.0. The suspension was centrifuged at 2,500 rpm for 5 min and the pellet was resuspended in 2 mL of PIV. After that 200 μL of the suspension was transferred to an Eppendorf and incubated in a bath at 50˚C for 30 min. Then, 200 μL of 1% PFGE agarose grade (Amresco) in the PIV buffer was added and mixture was poured into plug molds (Bio-Rad). After 30 min at 4˚C, the plugs were removed and incubated for 6 h at 37˚C in lysis solution (6 mM Tris-HCl, 1.0 M NaCl, 100 mM sodium EDTA, 0.5% Brij 58, 0.2% sodium deoxycholate, 0.5% sodium lauroyl sarcosine, 1 mg/mL lysozyme, 8-U/mL mutanolysin, pH 8), followed by an overnight incubation in a 50˚C water bath in ESP solution (0.5 M EDTA, 1% sodium lauroyl sarcosine, 0.2 mg/mL proteinase K, pH 8.0). The plugs were washed six times with TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6) for 10 min in a 50˚C shaker water bath each time. Then, plugs were stored in a fresh TE buffer at 4˚C. One third of a plug was incubated with 20 U of SmaI (BioLabs) in the enzyme restriction buffer for 2 h at 25˚C, and dispensed into wells of a 1% pulsed-field grade agarose gel (Amresco). The chromosomal digests were separated by PFGE, the first block with Table 1. Primer pairs used to detect lnuB, tetM and tetO.

Primer name
Primer sequence (5´-3´) (a) Annealing temperature product name and expected size

Results
Identification of the three isolates as S. equi subsp. zooepidemicus was confirmed by four different methods ( Table 2). API 20Strep, VITEK 2 and BBL-Crystal biochemical differentiation systems identified the three isolates as SEZ with high probability. MALDI-TOF MS showed the same result with a score between 2.28 and 2.42. Antibiotic susceptibility testing was assessed by the disc diffusion method and MIC determination by E-test. Isolates were susceptible to penicillin G, erythromycin, chloramphenicol, levofloxacin and vancomycin. Concerning tetracycline and clindamycin susceptibility, results differed among them. SEZ 559 and 594 exhibited intermediate resistance against tetracycline (MIC of 4 μg/mL both) while SEZ 567 was highly resistant (MIC of 32 μg/mL). PCR for the detection of tetM and tetO genes was performed on the three isolates. Only SEZ 567 was positive for tetM while in none of the isolates the tetO gene was amplified (S1 Fig). Analysis with clindamycin showed a borderline susceptibility by the disc diffusion method (20 mm and 19 mm, respectively) for SEZ 559 and SEZ 594 and a MIC of 0.50 μg/mL, interpreted as an intermediate resistance. SEZ 567, was categorized as resistant by both methods, although its MIC value was rather low (1 μg/mL). These results revealed that the three isolates presented the L phenotype, i.e. they were sensitive to erythromycin and resistant to clindamycin. Therefore, an analysis to identify the genetic basis of this phenotype was performed by PCR to detect the presence of the lnuB gene, as explained in Materials & Methods. An amplicon with the expected size (830 bp) was obtained from the three isolates (Fig 1). This positive result was achieved when the annealing temperature was reduced several degrees from that recommended by the enzyme producer (47˚C instead of 52˚C). Thus, an analysis of the conservation of the forward and reverse primers sequences was performed in data banks to hypothesize

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an explanation for this result. Indeed, while forward primer sequence was highly conserved among the lnuB releases, the region of the reverse primer appeared quite variable downstream the stop codon, which could explain the negative results under stringent conditions. On the contrary, for the reference strain S. agalactiae M6390, the PCR product invariably appeared as a strong band in all the conditions assayed. Amplicons from the three SEZ isolates were sequenced using primers lnuB-F and lnuB-R, and an identical 804 bp-lnuB sequence was found in all of them. It should be stressed that the first 6 bp and the final 14 bp of the gene are not corroborated since these sequences form part of the primers. The analysis in data banks revealed that the whole 804 bp sequence was the same as the lnuB one reported in many plasmids from E. faecalis and E. faecium, as well as in the chromosome of some S. aureus and Streptococcus spp., mainly S. suis and S. agalactiae. In addition, the same sequence recently appeared in a S. dysgalactiae DY107 genome release (AC: CP082206), i.e. from a species more closely related to S. equi than the remaining lnuB positive strains. Until now, no lnuB sequences have appeared in SEZ. Looking at the genetic environment of lnuB matches, this gene was found to be embedded in the lsa(E) multiple resistance gene cluster in most cases. In view of the susceptibility profile of our isolates, we particularly looked for a possible association between lnuB and tetracycline resistance genes. The analyses of data banks revealed that tetM, tetO and tetL (coding for one of the most common tetracycline efflux pump in gram positive clinical isolates [17]) are located in plasmids together with the lnuB determinant in some strains of E. faecalis, E. faecium and in one of S. aureus (AC: MK784778, MK465704, MN437484, MG957432, KY290886, HF586889, JX560992). In addition, a resistance gene cluster containing tetL was detected adjacent to the lsa(E) cluster in the chromosome of two S. agalactiae clinical isolates (AC: CP031556, MK102985), as well as the tetO gene in the chromosome of the S. dysgalactiae DY107 strain (AC: CP082206). Finally, the genetic relatedness of the three SEZ isolates was analyzed by PFGE and MLST. They all exhibited different pulsotypes, nevertheless, two of them, isolates SEZ 567 and SEZ

Discussion
In this study, three isolates of Streptococcus equi subsp. zooepidemicus-with no proven epidemiological link-were identified. This is the first report of human infections caused by this species in Uruguay. The isolates were recovered between 2018 and 2019 from elderly patients who developed invasive and severe infections (fatal meningitis, bacteremia, pneumonia). These findings correlate with previous observations pointing out SEZ as an uncommon but serious cause of human infections, particularly in elderly with underlying medical conditions or immunosuppression, and persons exposed to animals or animal products [3][4][5]36,37]. Group C streptococci meningitis is a rare type of infection, with approximately 50 cases reported, where SEZ appears as the most frequent causative agent [38][39][40]. Particularly in Latin-America, only three descriptions of meningitis caused by SEZ have been reported, one of them with a fatal outcome [40][41][42]. Thus, the meningitis caused by SEZ 559 is the fourth case identified in this region.
MLST and PFGE have been used in epidemiologic investigations of SEZ to determine the source of human infections, i.e. if the zoonosis resulted from direct contact with animals or from the consumption of dairy products [4,5,12,16,37,[43][44][45]. PubMLST global database of Streptococcus zooepidemicus currently includes 830 SEZ isolates (http://pubmlst.org/ szooepidemicus), most from Europe (77%). SEZ showed a wide genetic variation, i.e. in South America 23 isolates displayed 19 different ST and in North America 79 isolates displayed 55 different ST. Interestingly, two of our isolates share the same MLST, ST431. This ST was deposited in PubMLST database and corresponded to only one isolate from a horse infection in 2018 in Argentina (Bustos CP, personal communication). Taking into account that it was isolated in the same period of time and in the same region (our neighboring country, Argentina), an epidemiological relationship between this and our ST431 isolates could be suggested. Besides, SEZ 567 and SEZ 594 would be the first reports of human isolates with this sequence type. The other ST identified, ST72, was represented by five isolates in PubMLST, three were from human origin (UK and Brazil) and two from horses (UK and United Arab Emirates). We explored further the features of the Brazilian isolate because Brazil is also the other neighbor country to Uruguay. The above mentioned isolate was associated to a large outbreak of nephritis during 1997 and 1998, caused by consumption of unpasteurized cheese. The isolates were analyzed by PFGE and later one of them was assigned to ST72 after its genome was completely sequenced [4,11].
Regarding antibiotic susceptibility, a comparative analysis with previous published descriptions of human SEZ isolates that included antibiotic resistance profiles (around 20 reports) was carried out. Our results agree with previous descriptions: isolates were sensitive to penicillin G, erythromycin, chloramphenicol, levofloxacin and vancomycin [5,7,13,[40][41][42]46]. As for

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Streptococcus equi subsp. zooepidemicus lnuB positive tetracycline and clindamycin susceptibility, seven reports account for the resistance to one or both of these antibiotics [4,5,7,8,15,16,43]. Although data are very scarce, resistance to these two antibiotics tended to appear together in previous descriptions as it is found in our isolates [5,7]. Another important aspect to be mentioned is the low MIC values for clindamycin exhibited by the three isolates, i.e. two categorized as intermediate and one as resistant. As it was described before, MICs produced by the L phenotype are borderline or even below the breakpoints of resistance to clindamycin in streptococci and staphylococci strains [19,20,27,[47][48][49][50]. Indeed, the four studies of SEZ that included the MIC for clindamycin reported values of 0.50, 1, 1.5 and 4 μg/mL [4,7,15,49].
Clindamycin is recommended for use in combination with penicillin as therapy for invasive S. pyogenes infections due to its inhibition on toxin production [51]; the same treatment has been applied for some infections caused by SEZ [52,53]. Thus, our report of lnuB in S. equi together with similar reports on other species of streptococci, stress the importance of continuous surveillance of clindamycin susceptibility.
In this work, the genetic basis for clindamycin and tetracycline resistance phenotypes was analyzed for the three SEZ isolates. The lnuB gene was found in all of them, which could explain the clindamycin resistance phenotype. To the best of our knowledge, this would be the first description of a lnuB gene in the context of S. equi subsp. zooepidemicus, and more generally the first time that a lnu determinant was found in the species S. equi.
It has been described that lnuB gene forms part of the lsa(E) multiresistance gene cluster, which basically comprises the resistance genes aadE, spw, lsa(E) and lnuB, always flanked by transposon sequences. It was postulated that this gene cluster has arisen in Enterococcus -that is harbored in plasmids and contains erm(B) and aacA-aphD in some cases-and has disseminated in staphylococci and streptococci by horizontal gene transfer [19,25,54]. As for streptococci, this multiresistance gene cluster is located in the chromosome of strains of some species of this genus [19,26,27,55], including the closely related S. dysgalactiae DY107 strain. In one of these descriptions, the lsa(E) cluster was found in an integrative and conjugative element of some S. suis isolates, which could be experimentally transferred by conjugation [26]. In the case of S. pyogenes, a clone containing a genomic island in which the lnuB is embedded in a different genetic environment was reported; i.e. lnuB is adjacent to lsa(E) gene and to the aminoglycoside-streptothricin resistance cluster [ant(6)Ia-sat4-aph(3')III], but not to aadE and spw genes [28]. Concerning the possible association between lnuB and tet genes, some lnuB-lsa (E) positive strains of streptococci contained tetM, tetO and/or tetL in their chromosomes [19,26,55]. Particularly, tet determinants appeared in isolates from the two species most closely related to S. equi: tetM in the genome of an emm93.0 S. pyogenes strain and tetO in the chromosome of the S. dysgalactiae DY107 strain (AC: CP082206) [28]. Thus, the probable genetic linkage between lnuB and tet resistant genes should not be ruled out. In this sense, tetM was detected in one of the isolates, SEZ 567, which is in agreement with the MIC value reported for tetracycline. As to the remaining isolates, SEZ 559 and SEZ 594, their intermediate resistance against tetracycline suggests the possible presence of genes that encode efflux pumps such as tetL. Based on the above, we consider the possibility that the lnuB gene present in the three SEZ isolates could be located in an lsa(E) gene cluster near to a tet cluster, both of which might be part of a mobile genetic element acquired by horizontal gene transfer. More studies should be conducted to determine the genetic environment of the lnuB gene as well as its ability to be horizontally transferred from the SEZ isolates.
In conclusion, in a 16-year laboratory surveillance of streptococcal infections performed at the NRL, three independent SEZ infections were identified for the first time in Uruguay. The isolates presented the L phenotype and coherently harbored the lnuB gene. To the best of our knowledge, this is the first report of lnu gene in S. equi species, revealing active lateral spread of the lnuB gene, and most probably of the associated lsa(E) multiresistance platform, in a new bacterial host of the Streptococcus gender.